Cockayne syndrome group B protein is engaged in processing of DNA adducts of lipid peroxidation product trans-4-hydroxy-2-nonenal

Abstract

Cockayne syndrome complementation group B (CSB) protein is engaged in transcription-coupled repair (TCR) of UV induced DNA damage and its deficiency leads to progressive multisystem degeneration and premature aging. Here, we show that human CSB-deficient cells are hypersensitive to physiological concentrations (1–10 µM) of a lipid peroxidation product, trans-4-hydroxy-2-nonenal (HNE), and in response to HNE they develop a higher level of sister chromatid exchanges (SCEs) in comparison to the wild-type cells. HNE-DNA adducts block in vitro transcription by T7 RNA polymerase, as well as by HeLa cell-free extracts. Treatment of wild-type cells with 1–20 µM HNE causes dephosphorylation of the CSB protein, which stimulates its ATPase activity necessary for TCR. However, high HNE concentrations (100–200 µM) inhibit in vitro CSB ATPase activity as well as the transcription machinery in HeLa cell-free extracts. Cell lines expressing CSB protein mutated in different ATPase domains exhibit different sensitivities to HNE. The motif II mutant, which binds ATP, but is defective in ATP hydrolysis was as sensitive to HNE as CSB-null cells. In contrast, motif V mutant cells were as sensitive to HNE as were the cells bearing wild-type protein, while motif VI mutant cells showed intermediate sensitivity to HNE. These mutants exhibit decreased ATP binding, but retain residual ATPase activity. Homology modeling suggested that amino acids mutated in motifs II and VI are localized closer to the ATP binding site than amino acids mutated in ATPase motif V. These results suggest that HNE-DNA adducts are extremely toxic endogenous DNA lesion, and that their processing involves CSB. When these lesions are not removed from the transcribed DNA strand due to CSB gene mutation or CSB protein inactivation by high, pathological HNE concentrations, they may contribute to accelerated aging.

Damage of DNA and proteins by major lipid peroxidation products in genome stability

“…We have also shown that the presence of bulky HNE-DNA adducts in the template strongly inhibits transcription by T7 RNA polymerase, and HeLa cells extract in vitro [137].…”

“…HeLa cell-free extracts [137]. Inhibition of cellular repair capacity by LPO products may be an important, additional mechanism of pro-carcinogenic consequences of inflammation.…”

Cockayne syndrome pathogenesis: Lessons from mouse models

“…A commonality between these cells is that they all have a high lipid content and metabolism, suggesting a linkage between CS vulnerability and lipid metabolic pathways. One possibility is that CSB and other CS proteins are required for detoxification of specialized DNA lesions that arise from lipid metabolic pathways, such as lipid peroxidation products (Maddukuri et al, 2009). Alternatively, a commonality between oligodendrocytes and photoreceptor cells is that they have highly specialized cell compartments (myelin sheets, photoreceptor discs) that require the coordinated and balanced expression of multiple proteins.…”

“…A recent study revealed increased levels of 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine, which are substrates for endonuclease VIII-like (NEIL1) DNA glycosylase, in brain and kidney of Csb m/m mice, suggesting a role of CSB in the repair of these types of oxidative DNA damage (Muftuoglu et al, 2009). Other lesions that are potential substrates for CSB are DNA adducts of lipid peroxidation although these have not yet been examined in Csb m/m tissue (Maddukuri et al, 2009). Finally, recent data indicate that Csb m/m cells accumulate higher levels of oxidative DNA damage in mitochondria and display increased sensitivity to metabolic inhibition (Aamann et al, 2010;Kamenisch et al, 2010;Osenbroch et al, 2009), implying mitochondrial DNA damage in CS pathogenesis.…”

Defective resolution of pH2AX foci and enhanced DNA breakage in ionizing radiation‐treated cockayne syndrome B cells

“…Accordingly, recent evidence indicates that cs cells are hypersensitive to the killing effects of ionizing radiation (IR) and oxidative chemicals and accumulate oxidized purines (2) and pyrimidines (3), lesions that are substrates for DNA base excision repair (BER). The cs cells are further hypersensitive to lipid peroxidation products (4). An intriguing issue is whether CS proteins may exert a general function in response to oxidatively damaged DNA, taking part in the processing of DNA breaks.…”